The generation of genetically modified animals has spurred great advances in our understanding of various aspects of growth and development. Multiple technologies have used either injection into a two celled embryo followed by implantation into a pseudo pregnant mother, or using stem cell aggregation techniques to generate either knockout or knockdown mice. These experiments are expensive, labor-intensive, time-consuming and require several female mice to serve as donors for embryos and to serve as pseudo pregnant mothers for transplantation with a number of invasive surgical procedures.
Spermatogonial stem cells are responsible for the production of spermatozoa by spermatogenesis4 and hence an appropriate target for the germline modification. Earlier groups have generated transgenic mice by spermatogonial stem cell manipulation in vitro using either recombinant retroviruses or lentiviruses to infect spermatogonial stem cells in vitro and then transplant the cells into the testes of isogenic adult male mice, however, in some cases the recipient mice were unreceptive to the donor spermatogonial cells. Additionally, a loss of fertility has also been observed in some mice after in vivo transfection of testicular germ cells with retroviral constructs carrying a Iacz gene, and only 26% of the fertile males sired transgenic mice contributing to a poor success rate of 2.8%. In vitro manipulation of spermatogonial cells using lentiviral vectors followed by micro injection in testis increased the success rate to 6%. Recently, Majumdar and colleagues have generated transgenic mice by electroporation of an expression construct into the testes of adult male mice. While most of the fore founder mice were able to sire transgenic pups, the percentage of pups that were transgene positive is not known. Further, it was not clear whether germline transmission of the transgene was achieved.
All prior experiments involved manipulation of the spermatogonial cells in vitro followed by implantation into a donor. This is not a very efficient process and thus results in a low number of progeny that express the transgene in question. In practice this means that it sometimes takes one or two years to develop a transgenic mouse model which often retards scientific progress. While electroporation of DNA into the testes of the male mouse has avoided a number of these problems, the currently available data do not indicate whether the transgene is transmitted in the germ line. Therefore, there is a need for new technologies for generating transgenic mice that will address the issues raised above.